HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 39, 26634-26642, September 26, 2008

This Article Full Text Full Text (PDF) Author Profile Supplemental Data All Versions of this Article: 283/39/26634    most recent M802946200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cheng, G. Articles by Vierling, E. Search for Related Content PubMed PubMed Citation Articles by Cheng, G. Articles by Vierling, E. Related Collections Papers Of The Week Social Bookmarking                      What's this?

Insights into Small Heat Shock Protein and Substrate Structure during Chaperone Action Derived from Hydrogen/Deuterium Exchange and Mass Spectrometry^*

Guilong Cheng, Eman Basha, Vicki H. Wysocki, and Elizabeth Vierling¹

From the Departments of Chemistry and Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721

Small heat shock proteins (sHSPs) and the related -crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95–156 and 228–252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.

Received for publication, April 17, 2008 , and in revised form, June 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM42762 (to E. V.) and Grant GM051387 (to V. H. W.). This work was also supported by the 2005–2006 Pfizer Graduate Research Fellowship in Analytical Chemistry (to G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

This article was selected as a Paper of the Week.

¹ To whom correspondence should be addressed: 1007 E. Lowell St., University of Arizona, Tucson, AZ 85721. Tel.: 520-621-1601; Fax: 520-621-3709; E-mail: vierling{at}u.arizona.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?